CRISPR 101: Targeting RNA with Cas13a (C2c2)

Posted by Joel McDade on Sep 21, 2017 10:07:21 AM


CRISPR, and specifically Cas9 from S. Pyogenes (SpCas9), is truly an exceptional genome engineering tool. It is easy to use, functional in most species, and has many application (see a review of CRISPR applications here). That said, SpCas9 is not the only game in town, and several non-SpCas9 molecules have been characterized. Early research suggests that these molecules may circumvent the limitations associated with SpCas9 (see our blog entitled “Which Cas9 do I choose for my experiment”). A novel protein, Cas13a (previously referred to as C2c2), has several unique properties that make it particularly useful and further expand the CRISPR toolbox. This blog post will cover how Cas13a was identified, the structure and function of Cas13a with a focus on what makes this molecule unique, and the various applications of Cas13a.

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The Origins of Cas13a: An RNA Cleaving CRISPR Nuclease

Cas13a was originally identified by the Broad Institute in 2015 (Shmakov et al 2015).  Shmakov and colleagues were using Cas1, a gene commonly associated with CRISPR arrays and involved in spacer acquisition following infection, as a form of “bait” to identify new CRISPR-associated proteins within the bacterial genome.  From this analysis, they identified 53 potential candidate genes that fell into 3 categories based on the architecture of the CRISPR protein in question: C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, with the exception that they require both a tracrRNA and crRNA to cleave target DNA (Cpf1 requires only a crRNA for target recognition and cleavage).  C2c2, on the other hand, is unique in terms of structure and function, and will therefore be the focus of the remainder of this blog post.

Perhaps the biggest difference between Cas13a and Cas9 is that Cas13a binds and cleaves RNA rather than DNA substrates. In terms of structure, Cas13a shares no homology to the most commonly used CRISPR enzymes - Cas13a contains two HEPN domains, whereas Cas9 uses HNH and RuvC domains to cleave target DNA. The HEPN domains within Cas13a are essential for RNA cleavage, consistent with known roles for HEPN domains in other proteins.  Like Cas9, mutating key residues in the Cas13a molecule results in a “nuclease dead” Cas13a (dCas13a), that is capable of binding target RNA but lacks the ability to cleave the RNA target.

 Table 1: Comparison on Common CRISPR Enzymes

Name  Enzymatic Domains Guide RNA Target PAM (PFS) Cleavage Mechanism
Cas9 HNH, RuvC

TracrRNA, crRNA

DNA

5' NGG

Specific blunt ended DSB in target DNA

Cpf1

RuvC-like

crRNA DNA 5' TTN Specific DSB in target DNA with 5' overhangs
Cas13a (C2c2) 2x HEPN crRNA RNA 3' A, U, or C Specific RNA cleavage followed by non-specific RNAse activity

Cas13a Targeting with a Single crRNA

In the endogenous CRISPR/Cas9 system, Cas9 uses both a tracrRNA and crRNA to facilitate binding and cleavage of target DNA respectively.  Cas13a, on the other hand, uses only a short ~24 basepair crRNA that interacts with the Cas13a molecule through a uracil-rich stem loop and facilitates target binding and cleavage through a series of conformational changes in the Cas13a molecule.  Like Cas9, Cas13a tolerates single mismatches between the crRNA and target sequence, however cutting efficiency of Cas13a is reduced when 2 mismatches are present. The Protospacer Flanking Sequence (PFS) for Cas13a, which is analogous to the PAM sequence for Cas9, is located at the 3’ end of the spacer sequence and consists of a single A, U or C basepair.

One unique property of Cas13a is the fact that once Cas13a has recognized and cleaved its target RNA sequence as specified by the crRNA sequence, it adopts an enzymatically “active” state, where it will bind and cleave additional RNAs regardless of homology to the crRNA or presence of a PFS. This is in stark contrast to Cas9, which requires that each DNA target have high sequence identity to the spacer sequence and contain a PAM sequence just downstream of the sequence to be cleaved. This property is thought to activate programmed cell death or a dormant state for bacterial cells that have been infected with bacteriophage as to limit the spread of infection throughout the entire population. This property of Cas13a opens up the possibility of using Cas13a as a diagnostic tool, as discussed below.

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Potential Applications of Cas13a

Figure 1 Cas13a as Diagnostic Tool-01.png

So, what are the potential applications of Cas13a given what we know about its structure and function? For starters, Cas13a can be used to bind and cleave target RNAs, although its usefulness in this setting will likely be limited by the propensity of Cas13a to bind and cleave RNA non-specifically once the molecule has destroyed its target. Identifying variants or mutants of Cas13a that specifically cleave RNAs targeted by the crRNA but lack the ability to subsequently cleave nonspecific RNAs is undoubtedly an active area of research. The ability of dCas13a to bind target RNA without cleaving the molecule (like dCas9), suggests that dCas13a may be useful for isolating specific RNA sequences from a population (either enriching or depleting specific RNAs out of a pool of RNAs) or studying RNA processing in live cells using fluorophore tagged dCas13a. Finally (and perhaps most interestingly), a recent publication from the Zhang lab demonstrated that Cas13a’s propensity to cleave ALL RNAs after binding a user-defined RNA sequence could be used to detect single molecules of an RNA species with high specificity.  This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika virus, genotype human DNA and identify tumor mutations within cell-free genomic DNA.

In short, the Cas13a molecule brings a much anticipated element of diversity to the CRISPR toolbox and will continue to expand the applications of CRISPR.  It is worth noting that many of these applications have already been realized in the span of two short years since the original publication describing Cas13a!  We feel that the culture of sharing within the CRISPR community is an essential part of the rapid advancement seen in the CRISPR field and as always, we appreciate the opportunity to share these invaluable reagents with the scientific community.


References

1. Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353(6299) (2016):aaf5573. PubMed PMID: 27256883. PubMed Central PMCID: PMC5127784.

2. East-Seletsky, Alexandra, et al. "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection." Nature 538(7624) (2016):270-273. PubMed PMID: 27669025.

3. Gootenberg, Jonathan S., et al. "Nucleic acid detection with CRISPR-Cas13a/C2c2." Science (2017): eaam9321. PubMed PMID: 28408723. PubMed Central PMCID: PMC5526198.

4. East-Seletsky, Alexandra, et al. "RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes. " Mol Cell. 66(3) (2017):373-83. PubMed PMID: 28475872

5. Shmakov, Sergey, et al. "Discovery and functional characterization of diverse class 2 CRISPR-Cas systems." Molecular cell 60.3 (2015): 385-397. PubMed PMID: 26593719. PubMed Central PMCID: PMC4660269.

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